Biology subjects

Postovit, L.-M.

Publications and source records attributed to Postovit, L.-M..

12 recordsLinked to original sources

bioRxiv2026

Failure of Bacillus Calmette-Guerin Therapy in Patients with Bladder Cancer is Characterized by Immune Dysfunction Associated with Activator Protein 1

The standard-of-care for patients with higher-risk non-muscle invasive bladder cancer (NMIBC) after tumour resection is intravesical administration of Bacillus Calmette-Guerin (BCG). While this form of adjuvant immunotherapy has improved recurrence-free and progression-free survival, a large proportion of patients experience recurrences within a year of diagnosis. The reasons for this high rate of early recurrence following BCG therapy remain unclear; however, inadequate activation of systemic immunity may be a contributing factor. To address this, we analysed the transcriptomic and chromatin accessibility profiles of peripheral blood mononuclear cells obtained from patients with NMIBC at single-cell resolution before BCG immunotherapy and after five induction doses of BCG. Monocytes from patients who experienced disease recurrence within a year of initiation of BCG therapy (BCG non-responders) exhibited a pro-inflammatory phenotype consistent with age-related immunosenescence prior to BCG immunotherapy. Moreover, inflammation-associated pathways that were active before initiation of BCG therapy in the BCG non-responders were down-regulated after five instillations of BCG. In contrast, these pathways were quiescent before BCG therapy in patients who remained disease-free for at least a year but were markedly up-regulated after five doses of BCG. Genomic regions with accessible chromatin were enriched in activator protein 1 (AP-1) binding sequences in monocytes from BCG-non-responders prior to BCG therapy. AP-1 is a central regulator of the inflammatory phenotype associated with immunosenescence. Our findings indicate that a pre-existing state of innate immunosenescence underlies early disease recurrence following BCG. Patients unlikely to benefit from BCG may be offered alternative therapies early in their disease journey. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/723215v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@55b065org.highwire.dtl.DTLVardef@1afcea6org.highwire.dtl.DTLVardef@12833baorg.highwire.dtl.DTLVardef@5c22fc_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Cell State Chaos Underpins the Evolution of SMARCA4-Deficient Dedifferentiated Endometrial Cancer

Dedifferentiated endometrial carcinoma (DDEC) is a histologically unique cancer type, wherein well-differentiated regions lie adjacent to morphologically distinct, high-grade lesions that are histologically undifferentiated. Previous studies have determined that in nearly half of the cases dedifferentiation is associated with the genomic inactivation of SMARCA4, a catalytic subunit belonging to the SWI/SNF chromatin remodelling complex (SWI/SNF CRC), suggesting that SMARCA4 loss causes dedifferentiation. Herein, using gene editing, we reveal that when serially passaged in mice, SMARCA4-deficient endometrial cancer cells repeatably and predictably generate heterogeneous admixtures of differentiated and undifferentiated cells, resembling human DDEC. Surprisingly, despite this metamorphosis, SMARCA4 loss does not induce lineage plasticity nor reprogramming to a less differentiated fate. Rather, single-cell sequencing combined with barcoding demonstrated that SMARCA4 loss induces a dysregulated epigenome that allows cells to randomly move through cellular states that are otherwise shared with SMARCA4-expressing well-differentiated cancer cells. This finding was validated using a cohort of patient samples, such that epithelial fate markers (E-CADHERIN) can be detected in morphologically undifferentiated cells. Collectively, this work constitutes the first repeatable model of human dedifferentiated cancer and suggests that histological dedifferentiation is not due to the acquisition of a stem cell-like fate. Rather, undifferentiated tissue emerges due to epigenomic dysfunction concomitant with the arbitrary movement of cancer cells between cellular states.

cancer biology↗

bioRxiv2025

Isolation of Extracellular Vesicles from Minimal Volume Ascites Fluid Using Strong Anion Exchange Magnetic Beads

Ovarian cancer (OC) remains a leading cause of gynecologic cancer mortality due to late-stage diagnosis and limited early detection strategies. Ascites fluid, a pathological hallmark of OC, is a rich source of tumor-derived extracellular vesicles (EVs) that reflect the tumor microenvironment and hold promise for biomarker discovery. However, isolating EVs from minimal ascites volumes (<100 {micro}L) poses technical challenges using conventional methods like ultracentrifugation or size-exclusion chromatography (SEC). This study explores the application of strong anion exchange (SAX) magnetic beads (Mag-Net) for efficient EV isolation from as little as 2 {micro}L of ascites fluid from both murine models and a human patient with mucinous borderline tumor. We demonstrate that SAX achieves robust EV capture at 10{micro}l of input volume, enabling comprehensive proteomic profiling and single-EV surface-enhanced Raman spectroscopy (SERS) with a >2-fold increase in proteomic depth compared to raw ascites. Notably, this study was able to identify 1000 proteins not previously annotated in Vesiclepedia for OC-derived EVs, alongside distinct SERS signatures, highlighting the potential for multiomic analysis. Comparative analysis with UC revealed enhanced proteomic depth obtained with SAX beads, albeit we also observed differential detection of canonical markers (e.g., CD9, CD81) between input volumes of ascites fluid. These findings establish SAX as a scalable, low-input platform for EV-based biomarker discovery, paving the way for improved early detection and molecular insights into OC progression.

biochemistry↗

bioRxiv2024

Reduction in ETFDH expression optimizes cancer cell bioenergetics

Mitochondrial electron transport flavoprotein (ETF) insufficiency causes metabolic diseases known as a multiple acyl-CoA dehydrogenase deficiency (MADD). In contrast to muscle, ETFDH is a non-essential gene in acute lymphoblastic leukemia NALM-6 cells, and its expression is reduced across human cancers. ETF insufficiency caused by decreased ETFDH expression limits flexibility of OXPHOS fuel utilization but paradoxically increases cancer cell bioenergetics and accelerates neoplastic growth by activation of the mTORC1/BCL-6/4E-BP1 axis. Collectively, these findings reveal that while ETF insufficiency is rare and has detrimental effects in non-malignant tissues, it is common in neoplasia, where ETFDH downregulation leads to bioenergetic and signaling reprogramming that accelerate neoplastic growth.

cancer biology↗

bioRxiv2024

Mining the Proteome of Human Ovarian Cancer Extracellular Vesicles Using Thermolysin Proteolysis

This study investigates the utility of Thermolysin as a proteolytic enzyme to enhance the depth and coverage of proteomic analysis in ovarian cancer (OC) extracellular vesicles (EVs). EVs were isolated from OC cell lines and ascites fluid samples from women diagnosed with high-grade serous carcinoma. Proteins were digested using Thermolysin and Trypsin/LysC, followed by label-free data-dependent acquisition (DDA) and data-independent acquisition (DIA) mass spectrometry. The proteolytic efficiency, sequence coverage, peptide complexity, and proteomic depth were compared between Thermolysin and Trypsin/LysC digests. In silico analyses predicted theoretical benchmarks of these parameters using a core set of 22 proteins, and Gene set enrichment analyses (GSEA) highlighted the biological relevance of proteins identified throughout the study. Thermolysin digestion significantly increased the complexity or of peptide pools compared to Trypsin/LysC leading to limited peptide and protein identification, albeit total sequence coverage was increased through complementation to tryptic peptides. In both cell line and ascites EVs, Thermolysin identified unique proteins not detected by Trypsin/LysC that are known drivers of metastatic solid cancers, such as Ly6E. Offline strong cation exchange (SCX) fractionation improved proteomic depth and sequence coverage obtained with Thermolysin to generate a spectral library for DIA. Our DIA analysis of Thermolysin digests revealed the presence of NODAL in OC ascites EVs, a protein associated with poor clinical prognosis, which was not detected in Trypsin/LysC digests. The importance of NODAL was cross-validated in TGCA-OV and AOCS datasets by clinical cohorts by assessing RNA levels in solid tumors or ascites fluid, respectively. Collectively, we demonstrate that Thermolysin complements traditional enzymes like Trypsin/LysC to provide a more comprehensive proteomic landscape for biomarker discovery.

cancer biology↗

bioRxiv2023

MTOR modulation induces selective perturbations in histone methylation which influence the anti-proliferative effects of mTOR inhibitors

Emerging data suggest a significant cross-talk between metabolic and epigenetic programs. However, the relationship between the mechanistic target of rapamycin (mTOR) which is a pivotal regulator of cellular metabolism, and epigenetic modifications remains poorly understood. We thus explored the impact of modulating mTOR signaling on histone methylation, a well-known epigenetic modification. Our results showed that mTORC1 activation caused by abrogation of TSC2 increased H3K27me3 but not H3K4me3 or H3K9me3. This appeared to be mediated via the induction of EZH2 protein synthesis, downstream of 4EBPs. Surprisingly, mTOR inhibition also induced H3K27me3 independently of TSC2. This coincided with reduced EZH2 and increased EZH1 protein levels. Notably, the ability of mTOR inhibitors to induce H3K27me3 levels was positively correlated with their anti-proliferative effects. Collectively, our findings demonstrate that both activation and inhibition of mTOR selectively increase H3K27me3 by distinct mechanisms, whereby the ability of mTOR inhibitors to induce H3K27me3 influences their anti-proliferative effects. HighlightsO_LIParadoxically, both mTOR activation and inhibition induce H3K27me3. C_LIO_LIThe effect of mTOR inhibitors on H3K27me3 are not secondary to cell cycle arrest. C_LIO_LIH3K27me3 triggered by mTOR suppression coincides with perturbations in EZH1/2 ratio. C_LIO_LIH3K27me3 impacts on the anti-proliferative effects of mTOR inhibitors. C_LI

molecular biology↗

bioRxiv2023

Epigenetic coordination of transcriptional and translational programs in hypoxia

Adaptation to cellular stresses entails an incompletely understood coordination of transcriptional and post-transcriptional gene expression programs. Here, we quantified hypoxia-dependent transcriptomes, epigenomes, and translatomes in T47D breast cancer cells and H9 human embryonic stem cells. This revealed pervasive changes in transcription start site (TSS) selection associated with nucleosome repositioning and alterations in H3K4me3 distribution. Importantly, hypoxia-associated TSS switching was induced or reversed via pharmacological modulation of H3K4me3 in the absence of hypoxia, defining a role for H3K4me3 in TSS selection independent of HIF1-transcriptional programs. By remodelling 5UTRs, TSS switching selectively alters protein synthesis, including enhanced translation of mRNAs encoding pyruvate dehydrogenase kinase 1 (PDK1) that is essential for the metabolic adaptation to hypoxia. These results demonstrate a previously unappreciated mechanism of translational regulation during hypoxia driven by epigenetic reprogramming of the 5UTRome.

cancer biology↗

bioRxiv2020

Docosahexaenoic acid significantly reduces tumor cell growth in preclinical models of ovarian cancer

Despite recent advances in diagnosis and treatment, ovarian cancer (OC) is the most lethal gynecological malignancy and improving the efficacy of chemotherapy is of great interest. This study increases our understanding of how dietary intervention with docosahexaenoic acid (DHA)-a supplement proven safe for human consumption - enhances the anti-cancer effects of conventional chemotherapy. Our results demonstrated synergistic cell killing by DHA and carboplatin in OC cell lines. Furthermore, DHA supplementation alone and in combination with carboplatin significantly reduced OC growth in a high-grade serous OC patient-derived xenograft mouse model. Carboplatin administered intraperitoneally significantly reduced tumor growth in DHA-fed mice compared to mice on the control diet. Intravenous carboplatin administration in combination with DHA reduced tumor growth similarly to carboplatin or DHA monotherapies. The DHA-induced reduction in tumor growth in this model was associated with increased tumor necrosis and improved survival. As such, our findings provide a strong rationale to move to clinical trials that will determine whether DHA supplementation enhances the efficacy and tolerance of cytotoxic chemotherapy in patients with OC.

cancer biology↗

bioRxiv2019

Molecular profiling of lymphovascular invasive cancer cells in TNBC using G&T-seq

Lymphovascular invasion (LVI) is a critical step in the metastatic process but have received relatively little attention due to the technical challenges associated with their isolation. In this study, we used laser capture microdissection (LCM) to isolate 97 cancer cell clusters from pathological frozen sections within lymphatic vessels, primary tumor tissue, and axillary lymph nodes of a triple negative breast cancer (TNBC) patient. Simultaneous genome and transcriptome amplification and sequencing (G&T-seq) performed on these clusters permitted a comprehensive depiction of the genomic and transcriptional profiles of cancer cells associated with LVI. Combination phylogeny analysis pointed to three evolutionarily distinct pathways of tumor clone development and metastasis in this patient, each of which was associated with a unique mRNA signature, and correlated to disparate overall survival outcomes. Moreover, hub gene evaluation found extensive down regulation of ribosomal protein mRNA to be a potential marker of poor prognosis in breast cancer patients.

cancer biology↗

bioRxiv2019

Translational control of breast cancer plasticity

Plasticity of neoplasia, whereby cancer cells attain stem-cell-like properties, is required for disease progression and represents a major therapeutic challenge. We report that in breast cancer cells NANOG, SNAIL and NODAL transcripts manifest multiple isoforms characterized by different 5 Untranslated Regions (5UTRs), whereby translation of a subset of these isoforms is stimulated under hypoxia. This leads to accumulation of corresponding proteins which induce plasticity and \"fate-switching\" toward stem-cell like phenotypes. Surprisingly, we observed that mTOR inhibitors and chemotherapeutics induce translational activation of a subset of NANOG, SNAIL and NODAL mRNA isoforms akin to hypoxia, engendering stem cell-like phenotypes. Strikingly, these effects can be overcome with drugs that antagonize translational reprogramming caused by eIF2 phosphorylation (e.g. ISRIB). Collectively, our findings unravel a hitherto unappreciated mechanism of induction of plasticity of breast cancer cells, and provide a molecular basis for therapeutic strategies aimed at overcoming drug resistance and abrogating metastasis.

cancer biology↗

bioRxiv2018

Genetically regulated human NODAL splice variants are differentially post-transcriptionally processed and functionally distinct

NODAL is a morphogen essential for early embryonic development in vertebrates. Since much of our understanding of NODAL comes from model organisms, we aimed to directly assess post-transcriptional regulation of human NODAL with specific attention to a newly discovered human-specific NODAL splice variant. Selective depletion of the NODAL variant in human embryonic stem cells resulted in increased LIFR levels, while total NODAL knockdown resulted in a decrease of several markers of pluripotency. The NODAL variant did not transmit a canonical NODAL signal in zebrafish embryos, but may share some functional capability with canonical NODAL in cancer cells. At the protein level, disruption of disulfide bond formation dramatically enhanced proteolytic processing of NODAL. Disruption of NODAL N-glycosylation decreased its secretion but not extracellular stability, and a novel N-glycosylation in the NODAL variant contributed to enhanced secretion. Collectively, this work offers a direct and precise account of post-transcriptional regulation of human NODAL.

developmental biology↗

bioRxiv2018

Comprehensive characterization of transcript diversity at the human NODAL locus

NODAL, a morphogen belonging to the transforming growth factor beta (TG{beta}) superfamily, is essential during embryogenesis where it induces axis formation and left-right asymmetry. NODAL is also required for the maintenance of human embryonic stem cell pluripotency, and emerges in many cancer types concomitant with metastasis and therapy resistance. Several enhancer elements have been shown to regulate mouse Nodal expression and studies have delineated mechanisms by which mRNA splicing and translation of NODAL homologues are regulated in model organisms. However, little is known regarding the co-transcriptional and post-transcriptional processing of human NODAL. Herein, we describe hitherto unreported RNAs which are transcribed from the NODAL locus, including an antisense transcript, a circular transcript, and multiple splice variants. These transcripts demonstrate the complexity of NODAL expression and highlight the need to consider each NODAL variant when attempting to quantify or target this morphogen.

molecular biology↗